Figure 1: Elevation of cellular ROS levels in replicative aged HDFs through the activation of NADPH oxidase. (a) Increase in ROS with increasing PD of HDFs determined by ROS-sensitive fluorescent dyes. Various PDs of HDFs were stained with DCF-DA for 30 min and visualized with fluorescence microscopy. Senescent status was verified by in situ staining for SA-β-galactosidase. (b) ROS was measured by flow cytometry using DCFH-DA in various PDs of HDFs. (c) ROS in replicative aged HDFs (55 PD) under control conditions and in the presence of superoxide dismutase (SOD, 200 U/mL), rotenone (ROT, 10 mM), allopurinol (ALLO, 100 μM), diphenyleneiodonium (DPI, 100 μM), and apocynin (APO, 300 μM). (d) Increase in NADPH oxidase activity with increasing PDs of HDFs. NADPH oxidase activity was measured by lucigenin activity in the presence of 500 μM NADPH. (e) Multiple PDs of HDFs were analyzed by western blot for gp91phox, p67phox, and β-actin. Error bars, SD; in each group. , compared with 55 PD.